4th International Symposium on Emerging and Re-emerging Pig Diseases – Rome June 29th – July 2nd, 2003
IMMUNITY TO PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV): SYSTEMIC
AND LOCAL RESPONSES IN ACUTE AND PERSISTENT INFECTION
1 1 1 2 2
Z. Xiao , M. P. Murtaugh , C. R. Johnson , L. Batista , S. A. Dee
Departments of Veterinary Pathobiology and Clinical and Population Sciences, University of Minnesota, St. Paul USA
PRRS, immunity, T lymphocyte, interferon gamma, envelope glycoprotein
Porcine reproductive and respiratory syndrome virus Acute infection, as defined by presence of virus in blood, was
(PRRSV) causes a prolonged acute infection in pigs, in which similar in piglets and older animals, declining from near 100%
viremia lasts for 4-5 weeks, followed by a persistent infection on day 21 to nil between days 30-35. All lymphoid tissues were
of lymphoid tissues for several months. The course of PRRSV-positive at day 30.
infection contrasts to many other viral diseases of swine in
which the acute infection is resolved in one to two weeks and Antibody responses developed quickly, by day 14, to
sterilizing immunity is routinely achieved. Therefore, the nucleocapsid, and more slowly to envelope glycoprotein and
porcine immune response to PRRSV appears to be relatively ORF 1A proteases nsp1a/b and nsp2. Anti-GP5 titers were
ineffective. The humoral response to PRRSV has been not present until day 21, and peaked at day 28.
studied extensively (reviewed in 1), but the kinetics of Disappearance of virus from the circulation was most closely
antibody responses to individual PRRSV proteins is not associated with an increase in antibodies to GP5. The
known. Also, the cell-mediated immune response (CMI) is response of PRRSV-specific IFNg-secreting lymphocytes in
poorly described (1). We hypothesize that weak antigen- blood varied from nil to high (up to 436 positive cells per
specific immune responses are associated with prolonged million PBMC), was transient, and showed little correlation
viremia and persistent infection. To test the hypothesis, we with clearance of virus from the circulation. PRRSV-specific
expressed nonstructural proteins (nsp) 1, 2, and 4, envelope IFNg-secreting cells were detected in PBMC at day 14 post
glycoprotein (GP)5, and nucleocapsid (N) to assess PRRSV- infection (PI). Between days 21-28 the frequency of
specific humoral and cell-mediated responses in peripheral responding cells increased substantially and peaked at 28
blood and lymphoid tissues during acute and persistent days PI. Numbers declined thereafter until the end of the
infection. experiment at 52 days (Figure 1). Overall, PRRSV-specific
IFNg-secreting cells increased quickly in number, but also
One-hundred twenty female swine housed under commercial declined rapidly. In 4-month old animals, IFNg-secreting cells
conditions were infected at 4 months of age with PRRSV strain also were detected during acute infection, but peak levels
MN30100. A cohort of 11 animals and three uninfected controls over 1,000 positive cells per million PBMC were obtained at
were bled at 2-3 week intervals for 120 days for serum and approximately 70 days PI. As in piglets, these levels declined
whole blood. At three week intervals from day 30, three to a low level at 100 days PI.
infected animals were sacrificed and lymphocytes were
harvested from regional lymphoid tissues and bone marrow. In
Figure 1. Kinetics of IFNg ELISPOT response to PRRSV in
a second experiment, thirty-three 5-6 week pigs were
inoculated with Ingelvac PRRS ATP vaccine strain swine. Data are mean and standard error of 33 pigs per time
(Boehringer Ingelheim Vetmedica, St. Joseph MO) point through 38 days and 3 pigs per time point at days 42,
intramuscularly. EDTA-blood was collected by venipuncture 45, and 52.
at 7-10 day intervals for 52 days. Antibody responses were
measured in serum by ELISA to individual recombinant
proteins coated in microtiter plates. Cell mediated immune
responses were determined by interferon (IFN)g ELISPOT 100
since IFNg secretion by activated T lymphocytes, both in
helper and cytotoxic T cell subsets, is a hallmark of antiviral
immunity. Briefly, PBMC were isolated with lymphocyte 75
separation media (ICN Biomedicals, Irvine CA). Five x 10
cells in 50 µl RPMI 1640 complete medium were added to
duplicate wells of ELISPOT plates (Millipore, Bedford MA)
coated with anti-porcine IFNg monoclonal antibody P2G10 50
(BD Pharmingen, San Diego CA). Cells were stimulated with
PRRSV strain MN30100 in experiment 1, or VR2332 in
5 7 25
experiment 2, (2x10 TCID50) and 2.5 x 10 latex
microspheres coated with approximately 25 ng recombinant
nucleocapsid from strain VR2332 in 50 µl complete media.
Control wells received bovine serum albumin-coated beads or 0
phytohemagluttinin as negative and positive controls, 0 10 20 30 40 50 60
respectively. Cells were cultured in 5% CO2 at 37°C for 24 Days after Exposure
hours. Plates were washed 3 times with PBS, and 4 times Substantial variation was observed among piglets in the T cell
with PBS with 0.05% Tween-20. Captured IFNg was response to PRRSV. At peak responsiveness, on day 28,
detected with biotinylated anti-porcine IFNg antibody P2C11 four frequency categories were observed. A poorly
(BD Pharmingen) and streptavidin-HRP (Prozyme, San responding group with 4 of 33 animals (12%) varied from 3-
Leandro CA). Color was developed with 3-amino-9- 6
20 IFNg secreting cells/10 PBMC. A low responding group
ethylcarbazole (Sigma Chemical, St. Louis MO) and read on 6
with 28-60 responding cells/10 PBMC contained 13 of 33
an Immunospot Image analyzer (Cellular Technology, 6
pigs (39%), in which 11/13 had 32-50 cells/10 PBMC. T
Cleveland OH). Virus levels in serum and tissues were cells in 12 of 33 (36%) pigs secreted IFNg at an intermediate
determined by real time-PCR. Lymphocyte subpopulations 6
frequency of 72-114 cells/10 PBMC, and 4 highly responding
were characterized by FACS. 6
animals (12%) were >171 cells/10 PBMC, with the highest
4th International Symposium on Emerging and Re-emerging Pig Diseases – Rome June 29th – July 2nd, 2003
animal at 436 PRRSV-specific IFNg-secreting cells per 10 in PRRSV infection. In spite of an inefficient primary immune
PBMC. This variation among animals may be due to genetic response to PRRSV, protection against challenge with both
polymorphisms, which are important in host susceptibility to homologous and heterologous virus strains was substantial.
HIV (2) and chronic viral hepatitis infection (3). The This suggests that better immunological indicators of anti-
differences in T cell responsiveness to PRRSV at 28 days PRRSV protection remain to be discovered. The findings also
after infection were present throughout the period of acute indicate that a more robust response to the initial infection
infection. Differences in IFNg response were not associated with PRRSV might effect a stronger immune response with
with clearance of viremia or protection against challenge. All reduced duration of acute infection, limited persistence, and
animals cleared acute viremia and all animals were more effective sterilizing immunity. In support of this concept,
essentially free of lung lesions following challenge with highly danger signals that stimulate innate immunity have been
virulent field isolates. shown to elicit stronger or more rapid humoral and cell-
mediated immune responses to PRRSV (10).
The transient presence of PRRSV-specific T cells in blood
was not due to a redistribution of cells from peripheral Portions of the study were performed on blood samples and
circulation to lymph nodes. Lymphoid tissues, including tissues provided gratis by Michael Roof and Kelly Burkhardt,
spleen, inguinal lymph node, sternal lymph node, mesenteric Boehringer Ingelheim Vetmedica, Ames, Iowa. Animal care
lymph node and bone marrow, harbored PRRSV-specific and handling were provided by students and staff of Iowa
lymphocytes during acute and persistent infection. Cell State University Çollege of Veterinary Medicine under the
numbers varied by tissue and time, with an overall increase direction of Dr. Patrick Halbur, and students of the Swine
until 50-70 days PI, followed by a decrease to low levels at Disease Eradication Center, University of Minnesota. The
day 120 PI. Bone marrow showed the opposite trend. Cell authors deeply appreciate their assistance. Funds for the
frequencies were not correlated with virus load in the tissues. research were provided by grants from the Minnesota Rapid
Tonsil, the primarily tissue sampled in persistence studies, Response Fund and the University of Minnesota College of
had high levels of PRRSV but almost no responding Veterinary Medicine.
lymphocytes. PRRSV-specific lymphocytes were most
abundant in spleen, sternal lymph node, and bone marrow. References
FACS analysis of lymphocyte populations in these tissues 1. Murtaugh, M.P. Z. Xiao, and F. Zuckermann. 2002.
was unusual for spleen and bone marrow, in which CD3+ T Immunological responses of swine to porcine reproductive and
cells were approximately 80% CD8+ and <1% CD4+, and respiratory syndrome virus infection. Viral Immunol. 15:533-547.
2. Al Jabri, A.A. 2002. HLA and in vitro susceptibility to HIV. Mol.
approximately 10% were g/d T cells. This observation, in
combination with the report of Rodriguez Carreño et al. (4) 3. Thursz, M. 2001. Genetic susceptibility in chronic viral hepatitis.
showing that IFNg-secreting porcine lymphocytes are CD8+, Antiviral. Res. 52:113-116.
suggests that PRRSV-specific T cells are CD8+ cytotoxic T 4. Rodriguez-Carreño, L., L. Lopez-Fuertes, C. Revilla, A. Ezquerra,
lymphocytes. Interestingly, 2% of bone marrow lymphocytes F. Alonso, and J. Dominguez. 2002. Phenotypic characterization
+ dim of porcine IFN-g-producing lymphocytes by flow cytometry. J.
were g/d CD8 , a phenotype not previously associated with
anti-viral immunity. It was not determined if a portion of these Immunol. Methods 259:171-179.
5. Sopper, S., D. Nierwetberg, A. Halbach, U. Sauer, C. Scheller, C.
cells were specific for PRRSV peptides. Stahl-Hennig, K. Maetz-Rensing, F. Schaefer, T. Schneider, V.
Ter Meulen, and J.G. Mueller. 2002. Impact of simian
Viral infection induces the activation and clonal expansion of immunodeficiency virus (SIV) infection on lymphocyte numbers
antigen-specific helper and cytotoxic T cells. Effector T cells and T-cell turnover in different organs of rhesus monkeys. Blood.
further induce direct or indirect enhancement of cellular and 2002 Oct 10 [epub ahead of print]
humoral immune responses. Because IFNg production is 6. Even, C., R.R.R. Rowland, and P.G.W. Plagemann. 1995.
Cytotoxic T cells are elicited during acute infection of mice with
characteristic of a wide variety of porcine T cell subsets, but lactate dehydrogenase-elevating virus but disappear during the
not of B cells or macrophages (4), and because changes in chronic phase of infection. J. Virol. 69:5666-5676.
peripheral blood are representative of host response to 7. Ostrowski, M., J.A. Galeota, A.M. Jar, K.B. Platt, F.A. Osorio , and
pathogenic challenge (5), enumeration of IFNg-secreting T O.J. Lopez. 2002. Identification of neutralizing and
lymphocytes in PBMC provides a global measure of antigen- nonneutralizing epitopes in the porcine reproductive and
specific T cell response to PRRSV infection. Our findings respiratory syndrome virus GP5 ectodomain. J. Virol. 76:4241-
suggest that individual pigs vary greatly in the ability to 4250.
8. Bautista, E.M., P. Suarez, and T.W. Molitor. 1999. T cell
respond to PRRSV infection, and that pigs generally mount a
responses to the structural polypeptides of porcine reproductive
limited cell-mediated immune response to PRRSV. These and respiratory syndrome virus. Arch. Virol. 144:117-134.
factors would facilitate the establishment of persistent 9. Lopez Fuertes, L., N. Domenech, B. Alvarez, A. Ezquerra, J.
infections. Persistent infection is a characteristic feature of Dominguez, J.M. Castro, and F. Alonso. 1999. Analysis of
Arteriviruses, and may be due to the ability of Arteriviruses to cellular immune response in pigs recovered from porcine
infect without stimulating potent cytotoxic immune responses respiratory and reproductive syndrome infection. Virus Res.
as reported in lactate dehydrogenase elevating virus (6). 64:33-42.
However, the presence of IFNg-secreting lymphocytes in 10. Foss, D.L., M.J. Zilliox, W. Meier, F. Zuckermann, and M.P.
Murtaugh. 2002. Adjuvant danger signals increase the immune
regional lymphoid tissues in frequencies different from those response to porcine reproductive and respiratory syndrome virus.
in blood suggest that a full understanding of PRRSV Viral Immunol. 15:557-566.
persistence requires and examination of both circulating and
tissue-associated T cells.
Taken together, the findings are consistent with antibody-
dependent resolution of acute infection, consistent with the
hypothesis of Ostrowski et al. (7). The transient and variable
cell-mediated response that appears in the circulatory sytem,
i.e. blood and spleen (8, 9), may facilitate persistence of
PRRSV in lymphoid tissues. Tonsil, with a particularly high
ratio of viral antigen to specific T cells, may play a special role